| BackgroundMechanical trauma is a major medical and economic problem and a common injury in clinics. It can induce hemorrhagic shock, tissue injury and the release of circulating factors. After initial stabilization in the injured people, mechanical trauma still might be life-threatening. Though it has been proved that organ failure after trauma is a main cause of death in late injury patients, the mechanisms of the secondary tissue injuy is unclear. So it is critical to identify the organ injury after trauma and investigate the possible mechanisms of organ dysfunction, which would be helpful for seeking appropriate treatment to decrease the incidence of the secondary organ injuy associated with trauma.Due to the establishment and improvement of pre-hospital emergency care systems, the primary injury has been effectively controlled. However, the secondary injuy is becoming a potential killer threatened traumatic patient, because it is easily missed. Recent clinical reports have suggested that mechnical trauma induces myocardial infarction several days after trauma even in the absence of direct heart injury within 24h after trauma. However, the mechanisms responsible for this trauma-induced secondary heart injury have not been identified. So far, the lack of ideal animal models is one of the major problems that limiting deep study on the secondary heart injury.In our previous studies, we used Noble-Collip drum to establish trauma-induced secondary heart injury. The results showed that Electrocardiograph (ECG), mean arterial blood pressure (MABP) and cardiac function in vivo within 24h after trauma did not change significantly when the rats were subjected to a total of 200 revolutions at a rate of 40r/min, but cardiac function in vitro 24h after trauma obviously deceased. The results suggested that the trauma models directly caused the heart injury, but the heart still maintained normal function under the regulation of nerve and humoral factors. However, the pathophysiological significance of the direct heart injury need to be further explored. Firstly, it is unclear whether the mechanical trauma model could result in secondary heart injury induced by mechanical trauma, or lead to the increased sensitivity of heart tissue to negative stimulus. Secondly, it is uncertain that what the reason that causes the direct heart injury is.Many studies have shown that the factors led to cell death primaryly mainly include reactive oxygen species (ROS), nitric oxide (NO) and inflammatory factors in mechanical trauma. Necrosis and apoptosis are two major forms of cell death. A certain number of myocardial cell losses will cause cardiac dysfunction. To explore the mechanisms of myocardial cell loss after trauma would benefit for searching for the best treatment to prevent myocardial injury, and be helpful for the prevention and treatment of other organ damage. In the present study, we observed the possible mechanisms of myocardial cell loss after trauma through the use of in vitro and in vivo experimental models and pharmacological inhibitors treatment, which would supply experimental evidence for seeking the best treatment to prevent multiple organ failure after trauma.SECTION 1The establishment of mechanical trauma model induced secondary heart injuryObjective:To observe whether the established mechanical trauma models with Noble-Collip drum could result in secondary heart injury, which would provide research conditions for revealing the possible mechanisms of secondary heart injury.Methods:1. The preparation of mechanical trauma model According to our previous studies, adult healthy male Sprague Dawley rats weighing 180g-220g were randomly divided into sham trauma group and trauma group. The rats were anesthetized with chloral hydrate (400 mg/kg, i.p), placed into a Noble-Collip drum (diameter: 30.5cm) and were subjected to a total of 200 revolutions at a rate of 40 rpm. As the wheel was rotated, trauma rats were traumatized, while sham trauma rats were fixed on the drum with tape and could not fall from a height.2. The detection of rat ECG, MABP and cardiac function in vivo The rats were reanesthetized, fixed and connected with ECG. Then a PE-10 catheter filled with heparinized 0.9% NaCl solution was inserted into the left ventricle along the right carotid artery for recording ECG, MABP, LVSP, LVDP,±dP/dTmax with BL-410 biological signal processing system.3. The detection of rat cardiac function in vitro The rat was stunned and a midsternal thoracotomy was performed. Then the heart was excised immediately, trimmed in ice-cold Krebs-Henseleit and mounted onto a Langendorff heart perfusion apparatus. The pacing lead was placed into the right ventricle, and a latex balloon was inserted into the left ventricular cavity through the left atrium and connected to a pressure transducer. LVSP, LVDP,±dP/dTmax were recorded with BL-410 biological signal processing system.4.The establishment of rat myocardial ischemia/reperfusion (I/R) model The rats were reanesthetized and a mid neck incision was performed. After a tracheostomy was carried out, the animals were intubated and connected with ECG. Left thoracotomy was performed and the heart exposed through the fifth intercostal space. The pericardium was incised and a 6-0 silk suture was placed around the proximal portion of the left coronary artery, beneath the left atrial appendage. The ligature ends were passed through a small length of plastic tube to form a snare. For coronary artery occlusion, the snare was pressed onto the surface of the heart directly above the coronary artery and hemostat was applied to the snare. Ischemia was confirmed by the camponotus upward elevation of the ST segment. After 30 min of occlusion, the hemostat was removed and snare released for reperfusion, with the ligature left loose on the surface of the heart. Successful reperfusion was indicated by the restoration of normal rubor. In sham-operated rats, the same procedure was executed, without tightening the snare.5. The detection of creatine kinase isoenzyme MB (CK-MB) Creatine kinase isoenzyme MB (CK-MB) in rat serum was determined by ABC-ELISA method. After adding serum samples and other reagents according to the manual, set the wavelength of the microplate reader at 450nm to determine the optical density (OD) value. The levels of CK-MB were indicated by OD value of the well.6. Statistical analysis All calculations and the statistical analysis were performed using the statistical software program SPSS 15.0. All values are presented as means±SEM. Differences between two means were compared by Student's t test. All of the other values were analyzed by ANOVA followed by a Fisher least significant difference (post hoc) test for multiple comparisons. Probabilities of P<0.05 were considered statistically significant.Results:1. There was no obvious change in ECG, MABP within 24h after mechanic trauma When the rats were subjected to a total of 200 revolutions at a rate of 40r/min, there was no obvious change in ECG, MABP within 24h after mechanic trauma. And there was no obvious bleeding in rat visceral. The 24h survival rate was 100% (Fig. 3, 4, 5).2. There was no obvious change in cardiac function in vivo within 24h after mechanic trauma+dP/dTmax and -dP/dTmax reflect the left ventricular systolic function and diastolic function, respectively. Compared the cardiac function in vivo at all time points in the trauma group with the sham trauma group, there was no significant difference (P>0.05) (Fig. 6).3. The isolated cardiac function decreased 24h after mechanic trauma in ratsCompared with the sham trauma group, +dP/dTmax 24h after trauma was significant decreased [(3414±208) mmHg/sec vs. (4251±168) mmHg/sec, P<0.01], and -dP/dTmax was also markedly decreased [(-3301±458) mmHg/sec vs. (-5221±488) mmHg/sec, P<0.01] 24h after trauma (Fig. 7). While compared the isolated cardiac function at other time points in the trauma group with the sham trauma group, there was no significant difference (P>0.05) (Fig. 7).These results suggested that the established trauma models might be able to simulate mechnical trauma resulted in secondary heart injury.4. No obvious change was observed in cardiac function in vivo at 1week after mechanic traumaTo further observe the change of cardiac function at 1week after trauma in established traumatic models,±dP/dTmax was detected 1week after trauma (the common time point of secondary heart injury in clinic). The results showed that +dP/dTmax 1week after trauma was (5580±215) mmHg/sec. Compared with the sham trauma group, (5810±460) mmHg/sec, there was no significant difference (P>0.05). -dP/dTmax 1week after trauma was (-4390±87) mmHg/sec. Compared with the sham trauma group, (-4735±544) mmHg/sec, there was also no significant difference (P>0.05) (Fig. 8).5. Mechanical trauma increased the sensitivity of rat hearts to I/R injury at 1week after traumaTo further observe the susceptibility of the traumatic myocardium to I/R injury, the rats were subjected to I30min/R3h treatment 1week after trauma. And then cardiac function in vivo and the serum CK-MB level were detected. The results showed that compared with the sham traumatic I/R group, +dP/dTmax in traumatic I/R group was significant decreased [(2012±201) mmHg/sec vs. (3663±190) mmHg/sec, P<0.01], and -dP/dTmax in traumatic I/R group was also obviously decreased [(-1616±230) mmHg/sec vs. (-2602±246) mmHg/sec, P<0.01] (Fig. 9). Moreover, compared with the sham traumatic I/R group, the levels of serum CK-MB in traumatic I/R group was markedly increased [(4984±719) ng/ml vs. (2978±506) ng/ml, P<0.01)] (Fig. 10).Summary:In order to simulate trauma-induced secdondary heart injury and be consistent with the situation that only non-invasive examination can be used on clinic, the established trauma models should meet the following specification: non-invasive examination within 24h after trauma was normal, but there was occult cardiac injury that likely induced post-traumatic secdondary heart injury.In our previous studies, we found that ECG, MABP and cardiac function in vivo within 24h after trauma did not change significantly when the rats were subjected to a total of 200 revolutions at a rate of 40r/min, but cardiac function in vitro 24h after trauma obviously deceased. In the present study we firstly demonstrated mechanical trauma increased the sensitivity of rat heart to I/R injury 1week after trauma, even though cardiac function in vivo remained nomal. These results strongly suggested that this mechanical trauma models could simulate trauma-induced secdondary heart injury.SECTION 2The possible reasons responsible for mechanical trauma-induced secondary heart injuryObjective:To explore the possible reasons responsible for mechanical trauma-induced secondary heart injury through observation of the post-traumatic necrosis and apoptosis in myocardium within 24h.Methods:1. The detection of cardiac troponin I (cTnI) cTnI in rat serum was determined by ABC-ELISA method. After adding serum samples and other reagents according to the manual, set the wavelength of the microplate reader at 450nm to determine the optical density (OD) value. The levels of CK-MB were indicated by OD value of the well.2. The detection of myocardium apoptosis by terminal deoxyneucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay Myocardial tissues were embedded in a paraffin block and two slides at 4- to 5-μm thickness were cut from each tissue block. Immunohistochemical procedures for detecting apoptotic cardiomyocytes were performed by using an apoptosis detection kit according to the manufacturer's instructions. An additional staining was performed with monoclonal anti-αsarcomeric actin. This staining enables the identification of myocytes and, therefore, a distinction between myocyte nuclei and nuclei of other cells in cardiac tissue. After being rinsed with PBS, slides were coverslipped with mounting medium containing DAPI to permit total nuclei counting. Then the tissue slide was observed by laser scanning confocal microscopy (LSCM). 10 fields were selected randomly in each slide, and 200 cells were counted in one field. Total nuclei (DAPI staining, blue) and TUNEL positive nuclei (green) in each field were counted. The index of apoptosis (number of TUNEL-positive nuclei/total number of nuclei) was automatically calculated and exported to Microsoft Excel for further analysis. Results from different fields taken from the same animal were averaged and counted as one sample.3. The determination of myocardial Caspase-3, 8, 9, 12 activity In brief, after myocardial tissue was homogenized, centrifuged, the supernatants were collected, and protein concentrations were measured by the bicinchoninic acid (BCA) method. To each well of a 96-well plate, supernatant containing 200μg of protein was loaded and incubated with 25μg substrate. AFC was cleaved from DEVD and the free AFC was quantified by using microplate reader with a 400nm excitation filter and 505nm emission filter. The activities of Caspase-3, 8, 9, 12 were indicated by OD value of the well normalized with total protein concentration.4. The determination of protein concentration in myocardial tissue (BCA method) In brief, after adding the prepared working solutions, protein standards or the myocardial tissue supernatants into a 96-well plate, set the wavelength of the microplate reader at 512nm to determine the optical density (OD) value. The protein concentration of myocardial tissue was indicated by OD value of the well.5. The detection of creatine kinase isoenzyme MB (CK-MB) and cardiac function in vivo The method was the same as partâ… .6. Statistical analysis All calculations and the statistical analysis were performed using the statistical software program SPSS 15.0. All values are presented as means±SEM. Differences between two means were compared by Student's t test. All of the other values were analyzed by ANOVA followed by Fisher least significant difference (post hoc) test for multiple comparisons. Probabilities of P<0.05 were considered statistically significant.Results:1. Mechanical injury did not cause obvious myocardial necrosis in rats(1) There was no obvious change in serum CK-MB within 24h after mechanic traumaCK-MB is an index refelected myocardial necrosis. Compared the serum CK-MB at all time points in the trauma group with the sham trauma group, there was no significant difference (P>0.05) (Fig. 11).(2) No obvious change was observed in serum cTnI within 24h after mechanic traumacTnI is a highly sensitive and specific biomarker of myocardial necrosis. Compared the serum cTnI at all time points in the trauma group with the sham trauma group, there was no significant difference (P>0.05) (Fig. 12).These results suggested that the significantly reduced systolic and diastolic function 24h after trauma in isolated heart might not be the result of myocardial necrosis.2. Mechanical trauma increased cardiomyocytes apoptosis in rats.(1) The time course of cardiomyocytes apoptosis within 24h after mechanical trauma in ratsThe cardiomyocytes apoptosis was measured by TUNEL and Caspase-3 activity. The results showed that the myocardial apoptosis index in sham trauma group was (0.18%±0.05%), while it significantly increased 6h after trauma (6.71%±1.91%, P<0.01 vs. Sham) (Fig. 13). 12h after trauma it reached the peak, (14.42%±3.46%, P<0.01 vs. Sham), and it still remained at high level (8.12%±2.54%, P<0.01 vs. Sham) 24h after trauma. Caspase-3 activity markedly increased 6h after trauma [(51±4) vs. Sham (16.2±2.0), P<0.01]. 12h after trauma it reached the peak, (69±4, P<0.01 vs. Sham), and it still remained at high level (46±3, P<0.01 vs. Sham) 24h after trauma (Fig. 14).(2) The effect of Z-VAD-FMK (the broad-spectrum caspase inhibitor) on isolated cardiac function of traumatic ratsIn order to observe the role of myocardial apoptosis in isolated cardiac dysfunction induced by mechanical trauma, the trauma rats were treated with Z-VAD-FMK immediately after trauma. Cardiac function of traumatic rats was evaluated with isolated Langendorff heart perfusion. The left ventricular systolic function and diastolic function were assessed by +dP/dTmax and -dP/dTmax, respectively. The results showed that after Z-VAD-FMK treatment, +dP/dTmax 24h after trauma obviously increased compared with the trauma group [(4111±189) mmHg/sec vs. (3414±208) mmHg/sec, P<0.01]. After Z-VAD-FMK treatment, -dP/dTmax 24h after trauma was (-4997±351) mmHg/sec. Compared with the trauma group, (-3301±458) mmHg/sec, it was also significantly elevated (P<0.05) (Fig. 15). The results showed that Z-VAD-FMK could markedly improve the reduced cardiac function in vitro induced by trauma, which suggested that myocardial apoptosis played a very important role in the secondary heart injury induced by trauma.(3) The possible apoptotic pathways in cardiomyocytes within 24h after mechanical traumaTo observe the possible apoptotic pathways in rat cardiomyocytes within 24h after trauma, the activity of Caspase-8, Caspase-9 and Caspase-12 were detected. The results showed that Caspase-12 activity significantly increased 3h after trauma (66±8). Compared with the sham group (27±10), there was significant difference (P<0.01). 6h after trauma it reached the peak, (89±16, P<0.01 vs. Sham) , but 12h after trauma it significant decreased (P>0.05 vs. Sham)(Fig. 18). Caspase-8 activity significantly increased 24h after trauma (2312±648). Compared with the sham group (1449±296), there was significant difference (P<0.01)(Fig. 16). Caspase-9 activity also significantly increased 24h after trauma (875±460). Compared with the sham group (470±222), there was significant difference (P<0.01)(Fig. 17). The results suggested that in the early period of trauma, cardiomyocyte apoptosis was induced by activation the Caspase-12, an endoplasmic reticulum (ER)-specific caspase, following by activation of Caspase-8 (extrinsic pathway) and Caspase-9 (intrinsic pathway).Summary:1. The significantly reduced isolated systolic and diastolic function 24h after trauma might be related with cardiomyocyte apoptosis.2. In the early period of trauma, cardiomyocyte apoptosis was induced by activation the Caspase-12, an endoplasmic reticulum (ER)-specific caspase, following by activation of Caspase-8 (extrinsic pathway) and Caspase-9 (intrinsic pathway).SECTION 3The possible mechanisms of cardiomyocyte apoptosis induced by mechanical trauma.—The role of free radicals and inflammationObjective:1. To observe the production of reactive oxygen species, NO and the occurrence of inflammatory response after mechanical trauma.2. To explore the role of reactive oxygen species, NO and inflammation in cardiomyocyte apoptosis induced by mechanical trauma.Methods:1. The detection of totla NO (NOx) content in myocardial tissue (LDH method) After myocardial tissue was homogenized, centrifuged, the supernatants were collected. Then nitrate standards were prepared. After adding double distilled water, cardiac tissue supernatants, buffer and NADPH, nitrate reductase mixture, cofactor solution, LDH solution and Griess reagent R1 and R2 into a 96-well plate, set the wavelength of the microplate reader at 540nm or 550nm to determine the optical density (OD) value. The totla NO (NOx) content of myocardial tissue was indicated by OD value of the well.2. The detection of superoxide anion (O2ï¹’-) in myocardial tissue The generation of O2ï¹’- in myocardial tissues was detected by lucigenin-enhanced luminescence. In brief, cardiac tissues were placed into PBS solution containing lucigenin (0.25mM). RLU (relative light units) were determined by Monolight 2010 (BD, USA). The amount of O2ï¹’- in tissue samples was expressed as RLU per milligram of protein.3. The determination of nitrotyrosine (NT) content in rat myocardial tissue. Cardiac nitrotyrosine content was determined using an enzyme linked immunosorbent assay (ELISA) method. In brief, after the myocardial tissues were homogenized, centrifuged, the supernatants were collected and protein concentrations were determined by the BCA method. A nitrated protein solution was prepared for use as a standard. These standard samples, along with tissue samples from hearts, were applied to disposable sterile ELISA plates and incubated overnight with primary antibody. The secondary antibody was added, and the peroxidase reaction product was generated by using O-phenylenediamine dihydrochloride (OPD) solution. The optical density was measured at 490 nm with a microplate reader. The amount of nitrotyrosine content in tissue samples was expressed as nanomoles per gram of protein.4. The determination of nitric oxide synthase (NOS: iNOS, eNOS) expression by Western blot analysisIn brief, after the myocardial tissues were homogenized, centrifuged, the supernatants were collected and protein concentrations were determined by the BCA method. Equal amounts of protein (iNOS: 30μg protein/lane, eNOS: 60μg protein/lane) were electrophoresed on the 8% SDS-polyacrylamide gel and then electrophoretically transferred to a polyvinylidene di?uoride membrane. After blocking with blocking solution, the membrane was incubated with monoclonal antibody against either iNOS or eNOS. Following incubation with secondary antibody, the blot was developed with an ECL chemiluminescent detection kit and visualized with a Kodak Image Station 400. The blot densities were analyzed with Image-Pro Plus5.0 software.5. The determination of endothelial function of thoracic aorta Briefly, aortic rings were mounted onto hooks, suspended in organ chambers filled with K-H buffer and aerated with 95% O2 and 5% CO2 at 37°C, and connected to force transducers to record changes via a Powerlab data acquisition system. After equilibration for 60min at a preload of 1g, the rings were precontracted with norepinephrine. Once a stable contraction was achieved, the rings were exposed to cumulative concentrations of endothelium-dependent vasodilator (acetylcholine, ACh). After the cumulative response stabilized, the rings were washed and allowed to equilibrate to baseline. The procedure was then repeated with an endothelium-independent vasodilator (acidified NaNO2). A successful denudation of endothelial cells was confirmed by observing a lack of vasorelaxation to ACh, but a normal vasodilatation response to acidified NaNO2.6. The determination of myeloperoxidase (MPO) activity in myocardial tissue MPO activity was detected by colorimetric method. Myocardial tissues were homogenized (5% wt/vol) in homogenate media. In accordance with the instructions, once the proper reagents have been added to a 96-well plate, the OD values were recorded at 460 nm using a microplate reader.7. Immunohistochemical detection of MPO and intercellular adhesion molecule-1 (ICAM-1) Briefly, optimal cutting temperature compound-embedded tissues were cut into 6mm thickness and stained with primary antibody, secondary antibody. MPO was detected with alkaline phosphatase kit and ICAM-1 expression was detected with horseradish-peroxidase kit.8. The determination of protein concentration, Caspase-3 activity and TUNEL assay in myocardial tissue The method was the same as partâ…¡.9. Statistical analysisAll calculations and the statistical analysis were performed using the statistical software program SPSS 15.0. All values are presented as means±SEM. Differences between two means were compared by Student's t test. All of the other values were analyzed by ANOVA followed by a Fisher least significant difference (post hoc) test for multiple comparisons. Probabilities of P<0.05 were considered statistically significant. Vascular ring results were analyzed with a non-linear curve fitting by a Logistic equation with GrahPad Prism4.0 software.Results:1. The possible role of oxygen free radicals and nitrogen radicals in mechanical trauma-induced myocardial apoptosis(1) The change of myocardial O2ï¹’- levels within 24h after mechanical traumaCompared with the sham trauma group, the myocardial O2ï¹’- levels 6h after trauma obviously increased [(36.4±3.5) RLU/mg tissue vs. (12.7±1.8) RLU/mg tissue, P<0.01] (Fig. 19). 12h after trauma it still remained at high level, (29.6±4.2) RLU/mg tissue. Compared with the sham trauma group, there was significant difference (P<0.01) (Fig. 19).(2) The change of myocardial NOx content within 24h after mechanical traumaThe myocardial NOx content in sham trauma group was (2.12±1.1)μmol/g protein, while it increased to (9.5±1.9)μmol/g protein 6h after trauma. There was significant difference between the two groups (P<0.01) (Fig. 20). 12h after trauma it reached the peak, (17.2±2.3)μmol/g protein. Compared with the sham trauma group, there was significant difference (P<0.01) (Fig. 20).These results suggested that mechanical trauma resulted in the overproduction of O2ï¹’- and NO.(3) The change of post-traumatic myocardial NT content and the possible significanceIn many pathological situations, NO plays its toxic role by reacting with O2ï¹’- to generate peroxynitrite (ONOO-). ONOO- levels usually can be expressed by NT content. To investigate whether mechanical trauma could increase ONOO- content, NT levels were detected by ELISA method. The results showed that the myocardial NT content in sham trauma group was (0.78±0.26) nmol/g protein, while it increased to (12.2±1.1) nmol/g protein 12h after trauma. There was significant difference between the two groups (P<0.01) (Fig. 21).To explore the effects of ONOO- on NOx, NT and Caspase-3 activity in myocardial tissues, the trauma rats were treated with FeTMPyP (ONOO- decomposition catalyst) immediately after trauma. The results showed that treatment with FeTMPyP did not significantly alter the NOx content at 12h after trauma (Fig. 22). However, after FeTMPyP treatment, the myocardial NT content decreased to (3.2±1.0) nmol/g protein. Compared with trauma group (veichle control), there was significant difference (P<0.01) (Fig. 23). In addition, after FeTMPyP treatment, the myocardial Caspase-3 activity significantly decreased to (31.2±1.9). Compared with trauma group (veichle control), there was significant difference (P<0.01) (Fig. 24). Moreover, we accidentally discovered after FeTMPyP treatment, the myocardial NT content after trauma decreased about 70 percent.These results suggested that mechanical trauma increased ONOO- content, and FeTMPyP treatment could partially reduced cardiomyocyte apoptosis induced by mechanical trauma. Moreover, the result that FeTMPyP treatment could not completely recover NT content suggested there were other mechanisms that caused the overproduction of NT, in addition to the traditional view that increased ONOO- caused elevated NT.(4) The change of post-traumatic myocardial iNOS and eNOS and the possible significanceTo investigate the enzyme source of elevated NOx, iNOS and eNOS expression in myocardial tissue were detected by Western blot. The results showed that iNOS expression significantly increased 6h after trauma. Compared with the sham trauma group, there was significant difference (P<0.01) (Fig. 25). At 12h after trauma iNOS expression reached its peak, and it still remained at high level 24h after trauma. The myocafdial eNOS expression at each time point in trauma group had no significant difference compared with trauma group (P>0.05) (Fig. 26).To elaborate the effects of increased iNOS on NOx, NT and Caspase-3 activity in myocardial tissues, the trauma rats were treated with 1400W (a selective iNOS inhibitor) immediately after trauma. The results showed that administration of 1400W prevented the increase of the myocardial NOx, NT content and Caspase-3 activity. Respectively compared with NOx, NT and Caspase-3 activity of trauma group (veichle control), there was significant difference (P<0.01) (Fig. 27, 28, 29).These results suggested that mechanical trauma might increase NO production by upregulating iNOS expression, and then induced myocardial apoptosis.2. The role of inflammation response in mechanical trauma-induced myocardial apoptosis(1) The change of endothelial function of rat thoracic aorta after traumaThe inflammatory response is triggered by an early endothelial dysfunction. So firsty we observed the endothelial function of traumatized rat. The results showed that the maximal relaxation to cumulative ACh (10-9-10-5 mol/L) decreased from 85.89%±6.12% in sham trauma group to 61.55%±6.42% in trauma group (P<0.01) (Fig. 30A). There was no significant difference in acidified NaNO2-induced maximal relaxation between the two groups (P>0.01, Fig. 30B). The results suggested that mechanical trauma induced endothelial-dependent vasodilation dysfunction.(2) The change of myocardial ICAM-1 expression of traumatized ratICAM-1, as one of the most critical adhesion molecules, plays an important role in polymorphonuclear neutrophils (PMNs) adherence to the endothelium. The expression of ICAM-1 was determined by immunohistochemical staining. The results showed that ICAM-1 expression was markedly upregulated 6h after trauma in myocardial tissue obtained from traumatized rats (Fig. 31).(3) The activity and release of myocardial MPO in traumatized ratMPO, an enzyme occurring virtually exclusively in PMNs, was an index that has been shown to correlate closely with PMNs accumulation and infiltration in the heart. The activity and release of myocardial MPO in traumatized rats were detected by colorimetry and immunohistochemistry, respectively. The results showed that the myocardial MPO activity in sham trauma group was (0.58±0.24) U/g protein, while it increased to (2.91±0.36) U/g protein 6h after trauma. There was significant difference between the two groups (P<0.01) (Fig. 32A). 12h after trauma it reached the peak [(4.41±0.72) U/g protein vs. sham, P<0.01] (Fig. 32A). Moreover, compared with the sham trauma, MPO immunostaining was markedly increased in cardiac tissue from rats subjected to mechanical traumatic injury (Fig. 32B).These results suggested that mechanical trauma resulted in the basic pathologic changes of inflammation, including the damage of vascular endothelial, the increasing of ICAM-1 expression and MPO release, the raising of MPO activity.(4) The effect of MPO inhibitor on cardiomyocyte apoptosisTo further study the role of inflammation response in myocardial apoptosis induced by trauma, the trauma rats were treated with ABAH (a specific MPO inhibitor) immediately after trauma. The results showed that treatment with ABAH prevented the increase of the myocardial apoptosis index [(7.22%±1.24%) vs. trauma (veichle control, 14.42%±0.46%), P<0.01] (Fig. 33).The results suggested that ABAH could partially reduced mechanical trauma-induced cardiomyocyte apoptosis.(5) MPO was involved in the increased myocardial NT content It has been reported that there is ONOO- -independent protein nitration pathway besides ONOO-, in which MPO induced protein nitration is the most common. To observe whether the increased NT was related with MPO, the trauma rats were treated with ABAH. The results showed that after ABAH treatment, the myocardial NT content obviously decreased to (5.15±1.15) nmol/g protein. Compared with the trauma group (veichle control), there was significant difference (P<0.01) (Fig. 34).The results suggested that MPO might induce cardiomyocyte apoptosis by increasing NT content.Summary:1. Mechanical trauma significantly increased the levels of O2. -, NO, ONOO- and iNOS in myocardial tissues, and the cleavage of ONOO- and inhibition of iNOS significantly reduced cardiomyocyte apoptosis induced by mechanical trauma;2. Mechanical trauma resulted in the occurrence of cardiac inflammation response, and inhibition of MPO also significantly decreased cardiomyocyte apoptosis induced by mechanical trauma;3. Both ONOO- and MPO might result in myocardial apoptosis after trauma by increasing NT content. |
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